The biochemical basis for transport of plasma-membrane proteins from the rough endoplasmic reticulum (RER) to the cell-surface can be studied using a model system, Vesicular Stomatitus virus (VSV) infected Chinese Hamster Ovary (CHO) cells. an in vitro assay has recently been developed which measures the transport of a viral glycoprotein between the cisternae of the Golgi apparatus. Transport between the Golgi compartments in vitro requires an "intermediate". Extensive morphological evidence suggests that intracellular transport of proteins in vivo may occur by "transport vesicles". The relationship between these transport vesicles, and the "intermediate" observed in vitro will be explored. The immediate goals of the proposal are: (1) to purify and characterize the "transport intermediate" required for the transfer of proteins between the Golgi compartments. (2) to purify and characterize in vitro (a) enzymes which catalyze the formation of the intermediate from one Golgi-compartment, and (b) enzymes which catalyze the transfer of proteins from the intermediate to a second Golgi-compartment. (3) to develop a quantitative morphological measure of the in vitro transfer reaction using high-resolution electron microscope immunocytochemistry. (4) to isolate novel temperature-sensitive mutants of CHO cells which are pleiotropically defective in intracellular transport. These mutants will be used to genetically dissect the multi-component requirements of in vitro transport. Movement of proteins between cellular organelles must require both common transport components, and specific recognition signals. An integrated biochemical, genetic, and morphological approach using an experimentally versatile model system will provide a foundation to directly relate the events observed in vitro to those in vivo. Many medically important diseases result from defects in intracellular transport including lysosomal storage diseases, familial hypercholesterolemia, and cystic fibrosis. These studies will provide insight into the dynamics of membrane organization and assembly which regulates normal cellular metabolism.